Membrane proteins are critical components of all biological membranes, and can function as enzymes, receptors, channels and pumps. They are also very common in biological systems, as 20–40% of the genes found in the bacteria, archaea and eukaryotes code for membrane proteins (Wallin and von Heijne, Protein Sci, 7, 1029–38 (1998), Boyd, et al., Protein Sci, 7, 201–5 (1998), Gerstein, Proteins, 33, 518–34 (1998), Jones, FEBS Lett, 423, 281–5 (1998), Arkin, et al., Proteins, 28, 465–6 (1997)). Many clinically useful drugs, including the widely prescribed drugs, fluoxetine (Prozac™) and omeprazole (Prilosec™), interact with human membrane proteins. However, despite the abundance and importance of membrane proteins, this class of molecules is still only poorly understood at a structural level, mainly because of difficulties in growing crystals of membrane proteins suitable for analysis by x-ray crystallography (Garavito, et al., J Bioenerg Biomembr, 28, 13–27 (1996), Ostermeier and Michel, Curr Opin Struct Biol, 7, 697–701 (1997), Garavito, Curr Opin Biotechnol 9, 344–349 (1998)).
In order to understand the mechanism of action of a particular membrane protein, it is essential to know the three-dimensional structure of the molecule to a resolution that reveals its atomic structure. This is typically taken to be better than 0.3 nm resolution, and nearly all of the membrane protein structures that are known to this resolution have been determined by the technique of x-ray crystallography (Branden and Tooze, Introduction to Protein Structure, Garland Publishing Inc., New York (1998)). If the protein in question is medically important, knowledge of the 3-dimensional structure of the protein is a prerequisite for the development of new therapeutics using structure-based rational drug design methodologies (for example, see Klabunde, et al., Nature Structural Biology 7, 312–321 (2000)). The techniques used in the study of membrane protein crystals are very similar to those used for crystals of soluble proteins, and the main barrier to advancement in this field is the generation of diffraction-quality crystals.
The techniques used for the crystallization of membrane proteins are generally similar to the techniques used for the crystallization of soluble proteins, and include vapour diffusion, microdialysis and batch methods (A. McPherson, in “Crystallization of Biological Macromolecules”, Cold Spring Harbour Press (1998)). Typically, a purified, concentrated solution of protein is brought to the limit of its solubility over the course of days or weeks, resulting in either the formation of a protein precipitate or of protein crystals. Because precipitates are more often observed than crystals, numerous conditions are tested in these trials. The number of trials can vary in number from a few dozen to several thousand in attempts to find conditions resulting in crystal formation. The tested conditions can differ in pH, nature of added salts, concentration of the added salts, nature of the precipitant, concentration of the precipitant, temperature, and other factors (A. McPherson, in “Crystallization of Biological Macromolecules”, Cold Spring Harbour Press (1998)). In some instances, conditions producing suitable crystals for analysis by x-ray diffraction are not discovered even after extensive screening.
If the protein under consideration is an intrinsic membrane protein, the protein sample used in the crystallization trials is first purified and stabilized in a specific detergent in order to preserve the native conformation of the protein in the absence of a lipid bilayer (H. Michel, Trends Biochem. Sci. 8, 56–59 (1983), W. Kuhlbrandt, Quart. Rev. Biophysics 21, 429–477 (1988)). In most instances, a number of different detergents are tested for their ability to stabilize a particular membrane protein, and for their effect in the crystallization trials. Examples of detergents suitable for these purposes include the alkyl gylcoside detergents such as octyl β-D-glucopyranoside (OG, octyl glucoside) and dodecyl β-D-maltopyranoside (DDM, dodecyl maltoside) (Baron and Thompson, Biochim. Biophys. Acta 382, 276–285 (1975), Rosevear et al., Biochemistry 19, 4108–4115 (1980)), the polyoxyethylene alkyl ether detergents such as pentaethylene glycol monooctyl ether (C8E5) and octoethylene glycol monododecyl ether (C12E8) (Garavito and Rosenbusch, Meth. Enzymol. 125, 309–328 (1886), Victoria and Mahan, Biochim Biophys Acta 644, 226–232 (1981)), and the detergents described in U.S. Pat. No. 5,674,987, which are prepared from the reaction of a cycloalkyl aliphatic alcohol and a saccharide. Detergent-solubilized membrane proteins exist as protein-detergent complexes (PDC) in which a cluster of detergent molecules covers the surface of the protein that is normally exposed to the lipophilic core of the lipid bilayer. The hydrophobic portions of the detergent amphiphiles interact with the protein surfaces normally in contact with the lipid acyl chains, and thus mimic the normal lipid environment at the surface of the membrane protein. This micelle-like ring of detergent molecules surrounding the membrane protein is very dynamic and mobile, such that the surface properties of the PDC is in general poorly suited to the formation of well-ordered crystals (Crystallization of Membrane Proteins, H. Michel ed. CRC Press, Boca Raton, Fla. (1991)). This unfavorable effect is lessened in cases where the protein has large extramembranous domains, or with detergents that have small micellar volumes.
A number of techniques have been developed to address this difficulty in attempts to achieve membrane protein crystallization. For example, the formation of a complex with an antibody fragment has been used to increase the polar surface area of the Paracoccus denitrificans cytochrome oxidase, resulting in well-diffracting crystals (Ostermeier et al., Nat Struct Biol, 2, 842–6 (1995), Ostermeier et al., Proc Natl Acad Sci USA, 94, 10547–53 (1997)). Fusion proteins of the membrane protein lactose permease with soluble carrier domains have been made in attempts to achieve a similar result (Privé et al., Acta Cryst D50, 375–379 (1994), Privé and Kaback, J Bioenerg Biomembr 28, 29–34 (1996)). Bacteriorhodopsin (BR) has been crystallized from cubic lipid phases (Landau and Rosenbusch, Proc Natl Acad Sci USA, 93, 14532–5 (1996)) in a method that does not rely on detergents at all. However, few crystals suitable for structure determination have been produced by this method (Chiu, et al., Acta Crystallogr D56, 781–784 (2000)). A strategy to reduce the volume and dynamics of the detergent surface of the PDC has been proposed by Schafineister et al. (Science, 262, 734–8 (1993)). In this approach, amphipathic peptides have been used in the place of traditional detergents such as octyl glucoside. The peptides were designed such that the peptide would form an α-helix with one hydrophilic face and one hydrophobic face. The intention was that the hydrophobic surface was of the peptide would associate with the transmembrane surface of a membrane protein. Although the peptide used in this study could maintain some membrane proteins in a solubilized state for a few days, the proteins were not sufficiently stabilized for the purposes of crystallization. Because of their limited effectiveness as detergents, these peptides have not found general utility as tools for the study of membane proteins.
In the traditional detergents consisting of a polar head group and a linear alkyl tail, the length of the hydrocarbon moiety is an important factor in determining the ability of the detergent to preserve the native conformation of a solubilized membrane protein. Within the framework of a common head group, longer chain length detergents are generally more stabilizing towards membrane proteins, and are considered to be more “gentle”. The presumed mechanism for stabilization is that the longer chains are deemed to be more effective at masking the hydrophobic transmembrane surface of the membrane protein than the short chain detergents and are thus better mimics of the native membrane environment. However, longer chain detergents occupy a larger volume of the belt region of the PDC, a feature that is expected to reduce the probability of crystallization of the complex (Michel, 73–87 in “Crystallization of Membrane Proteins”, H. Michel, ed., CRC Press. Boca Raton, Fla. (1991)). Another factor affecting the choice of a particular detergent is the solubility of the detergent in water or buffer solutions. As the alkyl chain length increases in a series of detergents with a common head group, the overall solubility of the detergent decreases, eventually to levels making the detergent impractical for most uses. Thus, octyl glucoside is soluble to levels greater than 20% (w/v) in water, while decyl glucoside is soluble to only 0.1% (w/v) in similar conditions, and dodecyl glucoside is soluble only to 0.008% (w/v) (Anatrace Inc., Maumee Ohio 1999–2000 Catalogue). With a larger head group such as maltoside, the solubility of the long chain detergents increases, but solubility is still reduced to impractical levels with hexadecyl chain lengths or longer. Thus, within a series of traditional detergents, there is confict in the preferred length of the alkyl chain length. Long chains favor protein stability, and short chains are optimal for crystallization and detergent solubility. Since protein stability is a prime concern for crystallization trials, many membrane protein crystallization trials are carried out under sub-optimal conditions.
Thus, a major use of non-denaturing detergents is for the preservation of the biological function of a membrane protein in the absence of a lipid bilayer. These conditions are often encountered during the handling of membrane proteins, and in particular during the purification of membrane proteins, and during crystallization trials.
There is a need, thus, for a non-denaturing detergent which effectively mimics the membrane's lipid bilayer, is capable of solubilizing membrane proteins in such a way that the three-dimensional conformation is retained, and has features to enhance the probablility of crystallization of membrane proteins.